Infections caused by multi-drug resistance (MDR) Gram-negative pathogens are becoming increasingly difficult to treat, resulting in increased frequency of treatment failure and mortality. The increasing prevalence of MDR Gram-negative pathogens presents an unprecedented clinical challenge that requires innovative strategies for dealing with infections caused by these organisms. A major component of the MDR phenotype is the overexpression of efflux pumps, which bind to a broad spectrum of antibiotics and biocides and pump them out of the cell. To combat this problem, we will develop a novel class of drugs that inhibit the efflux pumps in MDR Gram-negative pathogens. These drugs will be used in combination with fluoroquinolone (FQ) or beta-lactam antibiotics to increase their clinical efficacy against MDR Gram-negative pathogens. In a previous screen, we identified a pyranopyridine (MBX-2319) that enhances the antibacterial activity of FQ and beta-lactam antibiotics against Escherichia coli, Shigella flexneri, Salmonella enterica, Enterobacter cloacae, and Klebsiella pneumoniae by inhibiting the major efflux pump in these organisms (AcrA-AcrB-TolC). When combined with ciprofloxacin, MBX-2319 increased bactericidal activity by >10000 fold, and reduces the MIC values of ciprofloxacin, levofloxacin and piperacillin by up to 8 fold against wild type, but not efflux deficient strains ( tolC and acrB) of E. coli. The hypothesis that MBX-2319 is an efflux pump inhibitor was supported by results obtained using a standard efflux assay (Hoechst 33342 accumulation assay) and in preliminary molecular docking studies with the AcrB crystal structure. The overall goal of this project is to develop a potent validated hit, the pyranopyridine (MBX-2319), into novel efflux pump inhibitors (EPI) to treat Gram-negative infections. These adjunctive therapies represent a significant improvement over single agent therapies, because they will provide the following benefits: 1) increased antibiotic efficacy at lower concentrations, and 2) decreased evolution of resistance. In Phase I, we will generate 4-6 lead compounds using medicinal chemistry to design and synthesize a library of analogs of MBX-2319 that will be evaluated to explore the structure activity relationships for the key properties required for a lead compound: potency, cytotoxicity, metabolic stability, and solubility Compounds that meet the rigorous criteria specified in the milestones will be designated as lead compounds. In Phase II, we will generate 2-3 optimized lead compounds that will be validated using in vivo models for toxicity, pharmacokinetics (PK), and efficacy in an animal (murine) model of infection. Compounds that meet the specified milestones will be designated as preclinical candidate compounds. The specific aims for Phase I are as follows: Aim 1. Generate a diverse library of MBX-2319 analogs for structure-activity-ADMET relationship studies. Aim 2. Evaluate MBX-2319 analogs for potency of antibiotic potentiation against Gram-negative pathogens. Aim 3. Prioritize analogs using in vitro cytotoxicity and ADME assays. Aim 4. Verify the mechanism of action and identify the molecular target of the MBX-2319 series of efflux pump inhibitors. PUBLIC HEALTH RELEVANCE: The tripartite efflux pump AcrAB-TolC is present in wild type Gram-negative bacteria and often over-expressed in MDR strains. Inhibition of this molecular machine will lead to increased antibiotic efficacy and decreased evolution of resistance. In the Phase I of this project, we propose a hit-to-lead development for MBX-2319, a novel small molecule, potent inhibitor of the AcrAB-TolC efflux pump, recently discovered in high throughput screening assays. In this project, we will explore the SAR of the pyranopyridine scaffold present in MBX 2319 and improve ADMET properties of the molecule. Lead compounds will be advanced to Phase II research. Our final goal is to develop a novel class of drugs that can be used in combination with fluoroquinolone (FQ), beta-lactam and/or other antibiotics to increase their clinical efficacy in treatment of Gram-negative infection diseases.